Personalized medicine is the application of genomic and molecular data to better target the delivery of health care to specific patients, facilitate the discovery and clinical testing of new products, and help determine a person's predisposition to a particular disease or condition.
On a technical level, personalized medicine depends on the identification and detection of proteins, genes and genetic variation (“biomarkers”) that play a role in a given disease. Rodland, Clin Biochem. 2004 July; 37(7):579-83. The presence or absence of certain biomarkers is then correlated with the incidence of a particular disease or disease predisposition. However, currently available methods for biomarker analysis are associated with long waiting periods, high cost and numerous technical hurdles.
The current standard for protein detection and/or quantification is based on immunoreactive detection (Western analysis). However, this technique requires the availability of an appropriately specific antibody. In addition, many antibodies only recognize proteins in an unfolded (denatured) form, cross-reactivity can be severely limiting, and quantification is generally relative.
The development of methods and instrumentation for automated, data-dependent electrospray ionization (ESI) tandem mass spectrometry (MS/MS) in conjunction with microcapillary liquid chromatography (LC) and database searching has significantly increased the sensitivity and speed of the identification of gel-separated proteins. Microcapillary LC-MS/MS has been used successfully for the large-scale identification of individual proteins directly from mixtures without gel electrophoretic separation (Link et al., 1999; Opitek et al., 1997). However, while these approaches accelerate protein identification, quantities of the analyzed proteins cannot be easily determined, and these methods have not been shown to substantially alleviate the dynamic range problem also encountered by the 2DE/MS/NIS approach. Therefore, low abundance proteins in complex samples are also difficult to analyze by the microcapillary LC/MS/MS method without their prior enrichment.
Protein ubiquitination is the one of the most common of all post-translational modifications. Ubiquitin is a highly conserved 76 amino acid protein which is linked to a protein target after a cascade of transfer reactions. Ubiquitin is activated through the formation of a thioester bond between its C-terminal glycine and the active site cysteine of the ubiquitin activating protein, E1 (Hershko, 1991, Trends Biochem. Sci. 16(7): 265-8). In subsequent trans-thiolation reactions, Ubiquitin is transferred to a cysteine residue on a ubiquitin conjugating enzyme, E2 (Hershko, et al., 1983, J. Biol. Chem. 267: 8807-8812). In conjunction with E3, a ubiquitin polypeptide ligase, E2 then transfers ubiquitin to a specific polypeptide target (see, e.g., Scheffner, et al., 1995, Nature 373(6509): 81-3), forming an isopeptide bond between the C-terminal glycine of ubiquitin and the 8-amino group of a lysine present in the target (See FIG. 1).
The covalent attachment of ubiquitin to cellular polypeptides, in most cases, marks them for degradation by a multi-polypeptide complex called a proteosome. The ubiquitinproteosome system is the principal mechanism for the turnover of short-lived polypeptides, including regulatory polypeptides (Weissman, 2001, Nat. Rev. Mol. Cell. Biol. 2: 169-78). Some known targets of ubiquitination include: cyclins, cyclin-dependent kinases (CDK's), NFKB, cystic fibrosis transduction receptor, p53, ornithine decarboxylase (ODC), 7-membrane spanning receptors, Cdc25 (phosphotyrosme phosphatase), Rb, Ga, c-Jun and c-Fos. Polypeptides sharing consensus sequences such as PEST sequences, destruction boxes, and F-boxes generally are also targets for ubiquitin-mediated degradation pathways (see, e.g., Rogers, et al., 1986, Science 234: 364-368; Yamano, et al., 1998, The EMBO Journal 17: 5670-5678; Bai, et al., 1996, Cell 86: 263-274).
Ubiquitin has been implicated in a number of cellular processes including: signal transduction, cell-cycle progression, receptor-mediated endocytosis, transcription, organelle biogenesis, spermatogenesis, response to cell stress, DNA repair, differentiation, programmed cell death, and immune responses (e.g., inflammation). Ubiquitin also has been implicated in the biogenesis of ribosomes, nucleosomes, peroxisomes and myofibrils. Thus, ubiquitin can function both as signal for polypeptide degradation and as a chaperone for promoting the formation of organelles (see, e.g., Fujimuro, et al., 1997, Eur. J. Biochem. 249: 427-433).
Deregulation of ubiquitination has been implicated in the pathogenesis of many different diseases. For example, abnormal accumulations of ubiquitinated species are found in patients with neurodegenerative diseases such as Alzheimer's as well as in patients with cell proliferative diseases, such as cancer (see, e.g., Hershko and Ciechanover, 1998, Annu Rev. Biochem. 67: 425-79; Layfield, et al., 2001, Neuropathol. Appl. Neurobiol. 27:171-9; Weissman, 1997, Immunology Today 18(4): 189).
While the importance of its biological role is well appreciated, the ubiquitin pathway is inherently difficult to study. Generally, studies of ubiquitination have focused on particular polypeptides. For example, site-directed mutagenesis has been used to evaluate critical amino acids which form the “destruction boxes”, or “D-boxes”, of cyclin, sites which are rapidly poly-ubiquitinated when cyclin is triggered for destruction. See, e.g., Yamano, et al., 1998, The EMBO Journal 17: 5670-5678; Amon et al., 1994, Cell 77: 1037-1050; Glotzer, et al., 1991, Nature 349: 132-138; King, et al., 1996, Mol. Biol. Cell 7:1343. Corsi, et al., 1997, J. Biol. Chem. 272(5): 2977-2883, which describe a Western blotting approach to identify ubiquitination sites. In this technique, crude radiolabeled a-spectrin fractions were ubiquitinated in vitro, digested with proteases, and electrophoresed on gels. Ubiquitinated peptides were identified by their differences in mass from peptides generated by digestion of non-ubiquitinated α-spectrin.
Although mass spectrometry offers a powerful tool for identifying ubiquitin substrates, a number of unresolved issues remain. Despite many advances, MS data is inherently biased toward more abundant substrates. The effects of ubiquitin epitope tags used to enriched ubiquinated proteins remain incompletely understood, including whether purification biases exist and whether ubiquitin pathway enzymes utilize tagged and wild-type ubiquitin with equal efficiency. It is also not clear if ubiquitin-binding proteins or ubiquitin antibodies may work efficiently as affinity reagents in order to lessen the need for epitope. Kirkpatrick et al., Nat Cell Biol. 2005 August; 7(8): 750-757.